Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
With the extension of the human life span, women spend one-third of their lives beyond the reproductive years. The transition into menopause, a normal process of aging, is associated with physical risks and psychological adjustments. It is critical to improve understanding of the these changes. However, there is a lack of diagnostic tools for monitoring the temporal stages in the history of menopause despite the importance of this transition period. There is no reliable test to determine how close a woman is to menopause. Clinical decisions for treatment of perimenopausal women today are based chiefly upon subjective symptoms rather than objective diagnostic tests.
There is a lack of adequate chemical markers for defining the menopausal state since neither serum gonadotropins, estradiol, nor inhibin A or B levels are adequate for diagnosis unless daily sampling is performed for prolonged periods of time (Burger, 1996;. Burger, et al., 1995; Burger, 1994a; Burger, 1994b; Hee, et al. 1993; Metcalf, 1988 ). A number of studies of women during the periovulatory period have indicated that the currently used biochemical markers of menopause are inadequate (Burger, 1996;. Burger, et al., 1995; Burger, 1994a; Burger, 1994b; Hee, et al. 1993; Metcalf, 1988; Santoro, et al. 1996). Gonadotropin levels fluctuate from postmenopausal cncentrations back down to levels found in normal, young cycling women (Burger, 1996; Burger, et al., 995; Burger, 1994a; Metcalf, 1988; Metcalf, et al. 1982; Metcalf and Donald, 1979). What appear to be normal ovulatory cycles may follow prolonged anovulatory periods coincident with postmenopausal concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) (Burger, 1996;. Burger, et al., 1995; Burger, 1994a; Burger, 1994b; Metcalf, 1988; Metcalf, and Donald, 1979; Metcalf, et al. 1981a). Some investigators declare that all current biochemical measurements have little predictive value during the menopausal transition because of the great variations in levels of steroids and gonadotropins. (Burger, 1996;. Burger, 1994a; Burger, 1994b; Hee, et al. 1993;. Metcalf, and Donald, 1979; Metcalf, et al. 1981b; Metcalf, 1979).
Although elevations in certain serum gonadotropin levels reflecting gametogenic failure usually occur several years before a decline in estrogen and irregular cycling begins, measurement of serum gonadotropin levels, estrogen, and inhibins A and B have limited value to the practicing physician. A reliable test is essential to differentiate a premenopausal woman from a woman very early in perimenopause or the laster from one in the middle of the transition; menopausal changes could be placed in relation to the stage of menopausal transition. This would help to resolve, or example, whether treatment for osteoporosis should begin much earlier or that hormone replacement therapy should begin at a different time rather than based on symptomatic discomfit. The present invention solves these problems by providing urinary-based immunoassay methods and assay kits
Human gonadotropins undergo metabolic transformations, which result in the presence of several smaller, structurally and immunologically related forms in the urine. A major form of urinary hCG-associated immunoreactivity is an epitope on a molecule smaller than heterodimeric hCG (Birken et al., 1996; O""Connor et al., 1994; Schroeder and Halter, 1983). This molecule has been identified as an hCG beta core fragment (hCGxcex2cf) (Birken et al., 1988; Blithe et al., 1988). In normal pregnancy, the core fragment constitutes a major mole fraction of urinary hCG excretion (Kato and Braunstein, 1988). Using polyclonal antisera raised against hCGxcex2cf, immunoreactive beta core like activity can be detected in both postmenopausal women and in the periovulatory period of the normal menstrual cycle (Iles et al., 1992; Neven et al., 1993). However, some immunoreactivity results from cross-reactivity with the polyclonal hCGxcex2cf antibodies. An hLH beta core fragment (hLHxcex2cf) has been isolated from human pituitaries and a panel of monclonal antibodies has been generated (Birken et al., 1993a; Kovalevskaya et al., 1995).
The corresponding urinary fragment is inferred from mass spectral and immunochemical analysis of chromatographically separated urinary forms. Physico-chemical characteristics, primarily mass spectral and chromatographic, indicate that the pituitary and urinary forms of hLHxcex2cf have a different structure, probably in the carbohydrate moieties. The carbohydrate moiety of the pituitary hLHxcex2cf resembles that of pituitary hLHxcex2 rather than displaying the degraded carbohydrate chains present in urinary hCGxcex2cf. The endogenous urinary core material is extremely stable to repeated freeze/thaw cycles and prolonged storage at 4xc2x0 C. or at room temperature. HLHxcex2cf cross-reaction with some hLH cr hLHxcex2 monoclonal antibodies may well interfere with the accurate estimation of the day of hLH surge when urinary tests are utilized. The expression of hLHxcex2cf in the urine of both reproductive and postreproductive age women and in men, is now characterized employing assays highly specific for the pituitary form of the fragment.
Analysis or the metabolites of the gonadotropins excreted into urine can help to distinguish between healthy and abnormal physiological states. For example, the hCG xcex2 core fragment (hCGxcex2cf) is present at high levels in the urine of normal pregnant women (Kato et al., 1988) but, also, occurs abnormally in the urine of nonpregnant patients with a variety of malignancies (O""Connor et al., 1988, Cole et al., 1988a,1988b,1990). Until now, it has not been possible to distinctly measure one of the fragments in the presence of the others. For example, the utility of the hCGxcex2cf molecule as a marker of malignancies in postmenopausal women has been compromised by the cross-reactions of antibodies elicited to the hCGxcex2cf with a molecule of similar structure and size (presumably the homologous fragment of hLH) excreted by normal postmenopausal women in their urine. Consequently, the high threshold measurement compromised the ability of hCGxcex2cf to serve as a cancer marker in this important patient population. Distinguishing hLHxcex2cf from an hCGxcex2cf, therefore, is of great value. A preponderance of hLHxcex2cf may indicate a normal state while a major mole fraction of the hCG fragment may be associated with malignancy (Birken et al., 1993b). The present invention provides a method to make such a distinction. Immunological analysis of the hLHxcex2cf in normal cycling women, as compared with infertile patients, may identify a metabolic marker associated with an abnormal state (i.e. an ovulatory cycles, polycystic ovarian disease). Antibodies to the hLHxcex2cf, isolated from pituitary extract, can also be used to measure such a molecule in urine.
Methods for specific immunometric assays which report the levels of expression of this new hLH molecular form, hHLxcex2cf, in men and women at different stages of their reproductive history are described herein. The present invention now makes it possible to evaluate the metabolism of hLH in premenopausal, perimenopausal and postmenopausal women and in men and to distinguish between normal and abnormal physiological states.
In addition, these methods to visualize LH fragment in plasma differentiates LH fragment derived directly from pituitary from that derived by peripheral cleavage of LH. hLHxcex2cf may circulate in plasma.
The methods described herein measure the stable metabolic products of LH which are excreted into urine usually at much higher concentrations than the parent hormones, themselves, are found in urine or blood. These assays do not use heterodimeric hormones which are unstable, unless supplemented by stabilizers such as glycerol, because they dissociate into their constituent, non-covalently bound subunits, especially under acid conditions or upon freeze thaw cycles. Urinary metabolic forms represent end-products of a degradative process. The forms explored have proven to be stable unlike the parent hormones which can dissociate into free subunits greatly complicating urinary measurements. Antibodies specific for hLH beta core fragment some of which are referred to in the present application, have been detailed in the related co-pending U.S. application Ser. No. 08/763,669 now U.S. Pat. No. 5,976,876 filed Dec. 11, 1996, the content of which is hereby incorporated by reference. In particular, related co-pending U.S. application Ser. No. 08/763,669 now U.S. Pat. No. 5,976,876 filed Dec. 11, 1996, describes monoclonal antibodies designated B505, B503 and B504 which are produced by the hybridoma cell lines accorded ATCC Accession Nos. 12000, 11999 and 12002 respectively and details methods for their production and use, which is hereby incorporated by reference.
This invention also provides monoclonal antibodies B503, B504 and B505 and the hybridoma cell lines for producing said antibodies. The hybridhybridoma cell lines were deposited pursuant to and in satisfaction of the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 on Dec. 11, 1995 under ATCC Designation Nos. 11999, 12002 and 12000, respectively. All restrictions upon public access to these deposits shall be removed upon the grant of a patent on this application and the deposit shall be replaced if viable samples cannot be made by the depository named hereinabove.
The present invention takes advantage of the natural metabolic processing of LH as a means of improving the diagnosis of women in perimenopause as well as to assess patterns of metabolites useful for monitoring estrogen replacement therapy.
The core fragment of hLHxcex2 is useful as a urinary marker for many different physiological states including disease, as markers of the state of senescence the ovary.
As used in this application hLH xcex2eta core fragment (hLHxcex2cf) means and includes a fragment of human luteinizing hormone (hLH) which is produced as a metabolize and which has been isolated from human pituitaries (Birken et al. 1993; Kovalevskaya et al., 1995) as well as related molecules and other metabolites of hLH which may be used as markers of menopause.
This invention provides a method for predicting the likely timing of the onset of menopause for a perimenopausal female subject by determining the amount of hLHxcex2cf in a sample from the subject comprising the steps of: (a) contacting a sample from the subject with an antibody which specifically binds to hLHxcex2cf without substantially cross-reacting with hLH, hLHxcex2 or hCGxcex2cf, under conditions permitting formation of a complex between the antibody and hLHxcex2cf; (b) measuring the amount of complex formed, so as to thereby determine the amount of hLHxcex2cf in the sample; and (c) comparing the amount of hLHxcex2cf in the subject""s sample determined in step (b) with either (i) the amount determined for known postmenopausal female subject or (ii) the amount determined for a sample from a known premenopausal female subject, wherein an amount of hLHxcex2cf in the sample similar to the amount of hLHxcex2cf in the known postmenopausal sample indicates temporal proximity to the onset of menopause, and an amount of hLHxcex2cf in the sample similar to the amount of hLHxcex2cf in the known premenopausal sample indicates temporal distance from the onset of menopause for the subject. As described herein amount is both concentration and pattern of measurement of concentrations in one or more consecutive urine specimens.
This invention further provides a method for predicting the likely timing of the onset of menopause for a perimenopausal female subject comprising the steps of: (a) contacting a urine sample from the subject with a capturing antibody which specifically binds to hLHxcex2cf without substantially cross-reacting with hLH, hLHxcex2 or hCGxcex2cf under conditions permitting binding of the antibody with any hLHxcex2cf present in the sample wherein the capturing antibody is bound to a matrix (b) separating hLHxcex2cf bound to the matrix bound capturing antibody from hLHxcex2cf not so bound; (c) contacting the hLHxcex2cf bound matrix to the capturing antibody with a second antibody which specifically binds to hLHxcex2cf that is bound to the capturing antibody without cross reacting with hLH, hLHxcex2 or hCGxcex2cf under conditions permitting binding of the second antibody to hLHxcex2cf bound to the capturing antibody; (d) measuring the amount of the second antibody bound to the hLHxcex2cf that is bound to the matrix bound capturing antibody so as to thereby determine the amount of hLHxcex2cf in the sample; and (e) comparing the amount of hLHxcex2cf in the subject""s sample determined in step (d) with either (i) the amount determined for a sample from a known postmenopausal female subject or (ii) the amount determined for a sample from a known premenopausal female subject, wherein an amount of hLHxcex2cf in the sample similar to amount of hLHxcex2cf in the known postmenopausal sample indicates temporal proximity to the onset of menopause, and the amount of hLHxcex2cf in the sample similar to the amount of hLHxcex2cf in the known premenopausal sample indicates temporal distance from the onset of menopause for the subject. As described herein amount is both concentration and pattern of measurement of concentrations in one or more consecutive urine specimens.
This invention also provides a method for determining the likely timing of the onset of menopause for a perimenopausal female subject comprising: (a) obtaining a series of samples from the female subject over a period of time; and (b) determining the amount of hLHxcex2cf in each of the samples, the presence of elevated levels of basal hLHxcex2cf in each of the samples indicating that the onset of menopause in the subject is likely to occur in the near future. As described herein amount is both concentration and pattern of measurement of concentrations in one or more consecutive urine specimens.
This invention further provides a method for assessing ovarian function in a subject comprising the steps of: (a) contacting a sample from a subject with an antibody which specifically binds to hLHxcex2cf without substantially cross-reacting with hLH, hLHxcex2 or hCGxcex2cf, under conditions permitting formation of a complex between the antibody and hLHxcex2cf; (b) measuring the amount of complex formed, so as to thereby determine the amount of molecule in the sample; and (c) comparing the amount of hLHxcex2cf in the subject""s sample determined in step (b) with either (i) the amount determined for a sample from a subject with normal ovarian function or (ii) the amount determined for a sample from a subject with abnormal ovarian function, wherein an amount of hLHxcex2cf in the sample similar to amount of hLHxcex2cf in the sample from subjects having normal ovarian function indicates normal ovarian function, and amounts of hLHxcex2cf in the sample similar to amounts of hLHxcex2cf having abnormal ovarian function indicates abnormal ovarian function for the subject. As described herein amount is both concentration and pattern of measurement of concentrations in one or more consecutive urine specimens.
This invention also provides a method for determining the efficacy of hormone replacement therapy in a perimenopausal female subject comprising the steps of: (a) contacting a sample from the subject with an antibody which specifically binds to hLHxcex2cf without substantially cross-reacting with hLH, hLHxcex2 or hCGxcex2cf, under conditions permitting formation of a complex between the antibody and hLHxcex2cf; (b) measuring the amount of complex formed, so as to thereby determine the amount of hLHxcex2cf; and (c) comparing the amount of hLHxcex2cf measured in step (b) with either (i) the amount determined for a sample from a subject taken prior to the commencement of therapy or (ii) the amount determined for a sample after a prior course of therapy (iii) the amount determined for a sample from a known premenopausal female subject or (iv) the amount determined for a sample from a known postmenopausal female, wherein differences in the amounts of hLHxcex2cf in the sample indicate efficacy of the hormone replacement therapy for the subject; amounts of hLHxcex2cf in the sample similar to amounts of hLHxcex2cf samples from known premenopausal subjects indicates efficacy of the hormone replacement therapy for the subject; amounts of hLHxcex2cf molecule in the sample similar to amounts of hLHxcex2cf in the sample from known postmenopausal subjects indicates lack of efficacy of the hormone replacement therapy for the subject. As described herein amount is both concentration and pattern of measurement of concentrations in one or more consecutive urine specimens.
Finally, this invention provides a diagnostic kit for predicting the likely timing of the onset of menopause for a perimenopausal female subject by determining the amount of hLHxcex2cf in a sample from the subject comprising: (a) a solid matrix to which an antibody which specifically binds to hLHxcex2cf without substantially cross-reacting with hLH, hLHxcex2 or hCGxcex2cf, under conditions permitting formation of a complex between the antibody and hLHxcex2cf is bound; and (b) a second antibody labeled with a detectable marker; and (c) reagents permitting the formation of a complex between the antibody and hLHxcex2cf. As described herein amount is both concentration and pattern of measurement of concentrations in one or more consecutive urine specimens.